Mirek Topolanek blamed the opposition for undermining the Czechs in Europe
The Czech PM says the collapse of his government will have "no impact" on the country's presidency of the EU.
Mirek Topolanek's centre-right coalition lost a no-confidence motion in parliament on Tuesday by one vote.
The government is half-way through its six-month term in the rotating presidency of the EU.
Attempts to form a new coalition are likely to precede any elections, but the Czechs will remain at the helm of EU affairs until June regardless.
Mr Topolanek was on European duty on Wednesday, addressing the European Parliament in Strasbourg, where he blamed the Czech opposition Social Democrats for undermining the Czech role in Europe.
"We have a case where the Social Democrats are obstructing the Czech Republic. It's a problem seen many times in the course of this presidency," he said.
"But don't be concerned, the situation will have no impact on the presidency," he added.
The government lost the vote in Prague after four rebel MPs voted with the opposition and Communists, giving them a majority of one.
Social Democrat leader Jiri Paroubek said ahead of the vote that the government could "complete the Czech EU presidency or its substantial part".
But Mr Topolanek has said he will step down, and ruled out the idea of a caretaker government until June, when the EU presidency passes to Sweden.
Foreign policy doubts
According to the constitution, Czech President Vaclav Klaus must decide who to choose to form a new administration. If three attempts to do so fail, early elections will be called.
The BBC's Rob Cameron in Prague says this surprise result, which threw observers completely off guard, could have far-reaching consequences beyond the Czech Republic's borders.
In addition to chairing the European Council, the Czech Republic is also in the middle of ratifying the Lisbon Treaty and is in talks with the United States on placing a radar base on Czech soil.
All these important foreign policy initiatives are now thrown into doubt, our correspondent adds.
The European Commission issued a statement expressing confidence that the Czech EU presidency would not be affected.
"The Commission has full trust that the national constitutional law allows for the Czech Republic to continue conducting the Council Presidency as effectively as it has done until now," it said.